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Ashley Callus swam in only one event in Sydney 2000, the 4 x 100 metres freestyle relay. In the final, he led off Chris Fydler, Michael Klim and Ian Thorpe to a thrilling victory over the United States on the first night of the swimming competition. Four years later at Athens 2004, he swam in the heats of the 50 and 100 metres and the sprint relay. Beijing 2008 saw him finish a close fourth in the 50 metres, behind Cielo Filho of Brazil and Leveaux and Bernard of France and, with Andrew Lauterstein, Eamon Sullivan and Matt Targett, win the bronze medal in the 4 x 100 metres freestyle relay, behind the Michael Phelps-led United States and France. At the 2001 world championships, Callus joined with Klim, Thorpe and Todd Pearson to win the 4 x 100 metres relay and inflict the first loss on the United States in this event at the swimming world titles. In 2002, he was the world short course champion in the 100 metres freestyle.